This invention generally relates to enzyme electrochemical sensors and, more particularly, to enzyme electrodes.
Enzyme electrodes are a class of devices that incorporate an enzyme as a catalyzing element on a conventional electrode. The enzyme is placed adjacent the electrode and catalyzes a reaction with a selected substance in which an electroactive species is formed (or consumed) and detected by the electrode to produce a signal functionally related to the amount of the selected substance adjacent the electrode. In potential medical applications of such sensors, small amounts of various substances contained in a body environment can be measured. Such substances may include glucose, urea, uric acid, triglycerides, amino acids, lactic acid, etc. Glucose concentrations are a particularly important indicator of various body conditions, e.g., diabetes, and glucose sensors may be combined with other devices to correct abnormal conditions.
By way of example, the enzyme glucose oxidase catalyzes the reaction of glucose with oxygen to produce gluconic acid and hydrogen peroxide. The presence of the hydrogen peroxide can be detected by an adjacent electrode and the amount of hydrogen peroxide can be determined, whereby the glucose concentration in the material adjacent the enzyme is then known. Conventionally, a selected enzyme is held adjacent the sample material by encapsulating the enzyme between membranes of a polymer suitable for passage of the material to be measured, by including the enzyme in the pore spaces of a suitable membrane, or by forming a membrane which incorporates the enzyme.
U.S. Pat. No. 4,415,666 issued Nov. 15, 1983, to D'Orazio et al., included herein by reference, teaches the use of cellulose acetate and copolymers of cellulose acetate to form a multilayer membrane, incorporating an enzyme in one layer. Numerous disadvantages of the prior art are discussed therein: cellophane membranes can pass interfering high molecular weight substances adjacent the enzyme; thin filter membranes can prevent the passage of interfering materials, but must be too thin to maintain electrode responsiveness to be of practical use; laminated structures require an enzyme adhesive to bond layers together and are subject to delamination; only low enzyme loadings are possible. The sensor taught by the '666 patent attempts to solve these problems by forming a two layer membrane of a cellulose acetate where the glucose oxidase is incorporated within one of the layers to immobilize the enzyme and to allow higher loadings of enzyme to be incorporated within the membrane. An outer layer of a higher density cellulose acetate is formed to contact the sample to preclude passage of interfering materials. The enzyme-containing cellulose acetate layer is formed directly on the high density layer to produce a substantially integral membrane. A membrane thickness of about 1-10 microns for the first layer and about 40-80 microns for the second layer is obtained. The subject membrane is placed in a polarograph having an electrolyte containing oxygen for generating hydrogen peroxide adjacent the cell electrode for sensing. The outer layer is also required to limit the flux of glucose adjacent the enzyme to preclude nonlinear signals arising from oxygen depletion in the membrane.
It would be desirable to provide the enzyme in a thin layer, i.e. less than about 10 microns thick, adjacent the electrode for a rapid response time for glucose concentration changes. The prior art electrodes also require an adjacent source of oxygen to maintain the enzyme reaction and, hence, are sensitive to local oxygen concentrations. It should also be noted that any material forming the sensor must be stable and biocompatible for possible in-vivo use. In addition, it is desirable to minimize interference from other oxidizable substances in a blood environment, such as ascorbic acid and uric acid.
The use of perfluorosulfonic acid polymers, and particularly Nafion (a trademark of Du Pont Company), as a protective membrane for use with glucose oxidase is taught by Harrison et al., "Characterization of Perfluorosulfonic Acid Polymer Coated Enzyme Electrodes and a Miniaturized Integrated Potentiostat for Glucose Analysis in Whole Blood," 60 Anal. Chem., No. 19, pp. 2002-2007 (Oct. 1, 1988), incorporated herein by reference. Enzyme-coated ion-sensitive field effect transistors (ISFET) were coated with Nafion to form a semipermeable membrane over the enzyme layer that reduced the sensitivity to O.sub.2 tension and provided satisfactory electrochemical performance, i.e., was semipermeable to glucose, protected the enzyme layer, was biocompatible, and obtained reproducible results. A device having a Nafion layer thickness of 1.7 microns was successfully operated in whole blood sample for about six days before the Nafion layer separated from a glass shroud around the electrode. The 1.7 micron thickness of Nafion provided a signal response at glucose concentrations as low as 1.2 mM. However, a linear response was obtained at glucose concentrations only up to 28 mM and the response time was 5-17 s. Harrison et al. also note problems with adhering the Nafion. Further, any openings in the Nafion cover would expose the enzyme to the material under test, resulting in degradation of the enzyme.
These and other problems of the prior art are addressed by the present invention and an improved enzyme electrochemical sensor is provided.
One object of the present invention is to provide a suitable enzyme adjacent the sensor electrode in a matrix in which the enzyme is not subject to degradation from the material under test.
Another object of the invention is to provide a thin enzyme layer having enhanced response times.
Yet another object is to provide an enzyme in a matrix which precludes interference from undesired oxidizable components in the material to be tested.
One other object of the invention is to provide an electrochemical sensor with a greatly reduced sensitivity to the oxygen tension adjacent the membrane.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.